ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcom... more ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neu-rodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling. Abstract A major problem in developmental neurotoxic-ity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of
Journal of Pharmacology and Experimental Therapeutics
Axonal swellings and neurofilamentous accumulations in the brain stem, spinal cord and peripheral... more Axonal swellings and neurofilamentous accumulations in the brain stem, spinal cord and peripheral nervous system are the most widely documented effects of exposure to 3,3'-iminodipropionitrile (IDPN). Evidence from morphological and functional studies, however, suggests that IDPN also may damage areas of the central nervous system above the level of the brain stem. To examine this possibility, we evaluated the astrocyte reaction to injury as an indirect means of detecting potential sites of IDPN-induced damage to the central nervous system. An immunoassay for the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP), was used to quantify gliosis. Rats were given IDPN (0-600 mg/kg/day i.p.) for 3 days. The concentration of GFAP in discrete brain regions was examined at postdosing times ranging from 3 days to 3 weeks. IDPN caused time-, dose- and region-dependent increases in GFAP; elevations were observed in the pons-medulla, midbrain, cerebral cortex and olfactory bulbs, but not in cerebellum, hypothalamus, hippocampus and striatum. Of these areas, cortex and olfactory bulbs showed the largest increases. Dissection of cortex into four subregions showed that the IDPN-induced increase in cortical GFAP was relatively uniform across this brain region. Application of the de Olmos cupric-silver degeneration stain to IDPN-treated tissue revealed intense argyrophilia in the glomerular layer of the olfactory bulbs and diffuse staining of axons in several regions of the cortex. The data indicate that IDPN is neurotoxic to the olfactory bulbs and cortex of the rat.
Pyrethroid insecticides have recently been purported to possess strong proconvulsant potential. T... more Pyrethroid insecticides have recently been purported to possess strong proconvulsant potential. The seizure-inducing properties of two pyrethroids were assessed by pentylenetetrazol (PTZ) seizure models (repeated ip, suprathreshold ip, and iv), and electrical kindling of the amygdala. The efficacy of po versus ip routes of deltamethrin administration was compared using iv-PTZ administration and tests of locomotor activity in a figure-eight maze. Both po and ip deltamethrin produced comparable decreases in motor activity indicating the effectiveness of both of these exposure routes on biological activity. The Type I pyrethroid, cismethrin (15 mg/kg, po), produced a 17% reduction in the threshold dosage of ip-PTZ required to induce a seizure, while delaying the onset of generalized seizure activity. The Type II pyrethroid, deltamethrin (10 mg/kg, po), failed to alter threshold or latency to seizure onset, but did increase seizure duration. No differences were revealed between po (0, 1...
Developmental neurotoxicity testing (DNT) is perceived by many stakeholders to be an area in crit... more Developmental neurotoxicity testing (DNT) is perceived by many stakeholders to be an area in critical need of alternative methods to current animal testing protocols and guidelines. An immediate goal is to develop test methods that are capable of screening large numbers of chemicals. This document provides recommendations for developing alternative DNT approaches that will generate the type of data required for evaluating and comparing predictive capacity and efficiency across test methods and laboratories. These recommendations were originally drafted to stimulate and focus discussions of alternative testing methods and models for DNT at the TestSmart DNT II meeting (http://caat.jhsph.edu/programs/workshops/dnt2.html) and this document reflects critical feedback from all stakeholders that participated in this meeting. The intent of this document is to serve as a catalyst for engaging the research community in the development of DNT alternatives and it is expected that these recomme...
Toxicological sciences : an official journal of the Society of Toxicology, Jan 13, 2015
We demonstrate a computational network model that integrates 18 in vitro, high-throughput screeni... more We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform ("assay interference"). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chem...
Triadimefon is a triazole fungicide that produces hyperactivity in both mice and rats similar to ... more Triadimefon is a triazole fungicide that produces hyperactivity in both mice and rats similar to that seen following administration of compounds with catecholaminergic activity (e.g., d-amphetamine). To determine whether the triadimefon-induced hyperactivity is due to an action on CNS catecholaminergic systems, we evaluated the effects of combined treatment of triadimefon with either the tyrosine hydroxlase inhibitor d,l-a-methyl-p-tyrosine methyl ester HCl
A modified method for the derivatization and determination of acrylamide as 2-bromopropenamide by... more A modified method for the derivatization and determination of acrylamide as 2-bromopropenamide by gas chromatography-electron-capture detection was developed and applied to serum and sciatic nerve from rats. The method was accurate and precise over the calibration range 2.24-7.47 micrograms/ml in serum diluted 1:125 and 4-122 micrograms/g in sciatic nerve homogenate (5 mg/ml). limits of detection were estimated to be 1200 ng/ml in undiluted serum and 3 micrograms/g in intact sciatic nerve. The use of less dilute samples to allow for lower limits of detection appears feasible. The time-course of acrylamide in serum and sciatic nerve was studied after acute dosing and indicated elimination half-lives of 1.8 and 2.0 h for serum and sciatic nerve, respectively. A dose-effect relationship was established for each matrix after acute dosing and the measured acrylamide concentrations in serum (microgram/ml) were approximately the same as in sciatic nerve (microgram/g).
A primary target of pyrethroid insecticides are the voltage-sensitive sodium channels (VSSCs). Ex... more A primary target of pyrethroid insecticides are the voltage-sensitive sodium channels (VSSCs). Expression of VSSCs in oocytes from Xenopus laevis is an experimental model used to study the effects of pyrethroids. A common assumption when utilizing this model is that media concentration is an accurate substitute for tissue dose. This assumption may not hold true for lipophilic chemicals. [3H]-deltamethrin (DLT) was used to test the hypothesis that media concentration is a good surrogate for tissue concentration. Accumulation of DLT (0.001-10 microM) in non-transfected oocytes exposed for 20 min was determined using liquid scintillation counting. The time course (1.0-180 min) of tissue accumulation of DLT (approximately 1.0 microM (0.50 ppm) in media) was also determined. Results demonstrate that tissue dose increases as a function of time with media concentration underestimating tissue dose at long incubation times (approximately 2.0-fold at 180 min) and overestimating tissue dose short incubation times (approximately 8.6-fold at 5 min). Tissue dose increases as a function of media concentration, with overestimation of tissue dose ranging from 1.5-fold at 0.0005 ppm to 4.1-fold at 5.0 ppm. These data suggest that media concentration does not accurately predict tissue dose at all times for a broad range of deltamethrin concentrations in X. laevis oocytes.
ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcom... more ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neu-rodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling. Abstract A major problem in developmental neurotoxic-ity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of
Journal of Pharmacology and Experimental Therapeutics
Axonal swellings and neurofilamentous accumulations in the brain stem, spinal cord and peripheral... more Axonal swellings and neurofilamentous accumulations in the brain stem, spinal cord and peripheral nervous system are the most widely documented effects of exposure to 3,3'-iminodipropionitrile (IDPN). Evidence from morphological and functional studies, however, suggests that IDPN also may damage areas of the central nervous system above the level of the brain stem. To examine this possibility, we evaluated the astrocyte reaction to injury as an indirect means of detecting potential sites of IDPN-induced damage to the central nervous system. An immunoassay for the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP), was used to quantify gliosis. Rats were given IDPN (0-600 mg/kg/day i.p.) for 3 days. The concentration of GFAP in discrete brain regions was examined at postdosing times ranging from 3 days to 3 weeks. IDPN caused time-, dose- and region-dependent increases in GFAP; elevations were observed in the pons-medulla, midbrain, cerebral cortex and olfactory bulbs, but not in cerebellum, hypothalamus, hippocampus and striatum. Of these areas, cortex and olfactory bulbs showed the largest increases. Dissection of cortex into four subregions showed that the IDPN-induced increase in cortical GFAP was relatively uniform across this brain region. Application of the de Olmos cupric-silver degeneration stain to IDPN-treated tissue revealed intense argyrophilia in the glomerular layer of the olfactory bulbs and diffuse staining of axons in several regions of the cortex. The data indicate that IDPN is neurotoxic to the olfactory bulbs and cortex of the rat.
Pyrethroid insecticides have recently been purported to possess strong proconvulsant potential. T... more Pyrethroid insecticides have recently been purported to possess strong proconvulsant potential. The seizure-inducing properties of two pyrethroids were assessed by pentylenetetrazol (PTZ) seizure models (repeated ip, suprathreshold ip, and iv), and electrical kindling of the amygdala. The efficacy of po versus ip routes of deltamethrin administration was compared using iv-PTZ administration and tests of locomotor activity in a figure-eight maze. Both po and ip deltamethrin produced comparable decreases in motor activity indicating the effectiveness of both of these exposure routes on biological activity. The Type I pyrethroid, cismethrin (15 mg/kg, po), produced a 17% reduction in the threshold dosage of ip-PTZ required to induce a seizure, while delaying the onset of generalized seizure activity. The Type II pyrethroid, deltamethrin (10 mg/kg, po), failed to alter threshold or latency to seizure onset, but did increase seizure duration. No differences were revealed between po (0, 1...
Developmental neurotoxicity testing (DNT) is perceived by many stakeholders to be an area in crit... more Developmental neurotoxicity testing (DNT) is perceived by many stakeholders to be an area in critical need of alternative methods to current animal testing protocols and guidelines. An immediate goal is to develop test methods that are capable of screening large numbers of chemicals. This document provides recommendations for developing alternative DNT approaches that will generate the type of data required for evaluating and comparing predictive capacity and efficiency across test methods and laboratories. These recommendations were originally drafted to stimulate and focus discussions of alternative testing methods and models for DNT at the TestSmart DNT II meeting (http://caat.jhsph.edu/programs/workshops/dnt2.html) and this document reflects critical feedback from all stakeholders that participated in this meeting. The intent of this document is to serve as a catalyst for engaging the research community in the development of DNT alternatives and it is expected that these recomme...
Toxicological sciences : an official journal of the Society of Toxicology, Jan 13, 2015
We demonstrate a computational network model that integrates 18 in vitro, high-throughput screeni... more We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform ("assay interference"). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chem...
Triadimefon is a triazole fungicide that produces hyperactivity in both mice and rats similar to ... more Triadimefon is a triazole fungicide that produces hyperactivity in both mice and rats similar to that seen following administration of compounds with catecholaminergic activity (e.g., d-amphetamine). To determine whether the triadimefon-induced hyperactivity is due to an action on CNS catecholaminergic systems, we evaluated the effects of combined treatment of triadimefon with either the tyrosine hydroxlase inhibitor d,l-a-methyl-p-tyrosine methyl ester HCl
A modified method for the derivatization and determination of acrylamide as 2-bromopropenamide by... more A modified method for the derivatization and determination of acrylamide as 2-bromopropenamide by gas chromatography-electron-capture detection was developed and applied to serum and sciatic nerve from rats. The method was accurate and precise over the calibration range 2.24-7.47 micrograms/ml in serum diluted 1:125 and 4-122 micrograms/g in sciatic nerve homogenate (5 mg/ml). limits of detection were estimated to be 1200 ng/ml in undiluted serum and 3 micrograms/g in intact sciatic nerve. The use of less dilute samples to allow for lower limits of detection appears feasible. The time-course of acrylamide in serum and sciatic nerve was studied after acute dosing and indicated elimination half-lives of 1.8 and 2.0 h for serum and sciatic nerve, respectively. A dose-effect relationship was established for each matrix after acute dosing and the measured acrylamide concentrations in serum (microgram/ml) were approximately the same as in sciatic nerve (microgram/g).
A primary target of pyrethroid insecticides are the voltage-sensitive sodium channels (VSSCs). Ex... more A primary target of pyrethroid insecticides are the voltage-sensitive sodium channels (VSSCs). Expression of VSSCs in oocytes from Xenopus laevis is an experimental model used to study the effects of pyrethroids. A common assumption when utilizing this model is that media concentration is an accurate substitute for tissue dose. This assumption may not hold true for lipophilic chemicals. [3H]-deltamethrin (DLT) was used to test the hypothesis that media concentration is a good surrogate for tissue concentration. Accumulation of DLT (0.001-10 microM) in non-transfected oocytes exposed for 20 min was determined using liquid scintillation counting. The time course (1.0-180 min) of tissue accumulation of DLT (approximately 1.0 microM (0.50 ppm) in media) was also determined. Results demonstrate that tissue dose increases as a function of time with media concentration underestimating tissue dose at long incubation times (approximately 2.0-fold at 180 min) and overestimating tissue dose short incubation times (approximately 8.6-fold at 5 min). Tissue dose increases as a function of media concentration, with overestimation of tissue dose ranging from 1.5-fold at 0.0005 ppm to 4.1-fold at 5.0 ppm. These data suggest that media concentration does not accurately predict tissue dose at all times for a broad range of deltamethrin concentrations in X. laevis oocytes.
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